The aim of biotechnological studies on plants is the preparation of plants having improved properties, for example to increase agricultural productivity. The preparation of transgenic plants is a fundamental technique in plant biotechnology and thus an indispensable prerequisite for basic research on plants in order for the preparation of plants having improved novel properties for agriculture, for improving the quality of foodstuffs or for the production of particular chemicals or pharmaceuticals (Dunwell J M, J Exp Bot. 2000; 51 Spec No: 487-96). The natural defense mechanisms of the plant, for example against pathogens, are often inadequate. The introduction of foreign genes from plants, animals or microbial sources can enhance the defense. Examples are the protection against insects feeding on tobacco by expression of the Bacillus thuringiensis endotoxin under the control of the 35 S CaMV promoter (Vaeck et al. (1987) Nature 328:33-37) and the protection of tobacco against fungal infection by expression of a chitinase from beans under the control of the CaMV promoter (Broglie et al. (1991) Science 254:1194-1197). It is furthermore possible to achieve resistance to herbicides by introducing foreign genes, thereby optimizing the cultivation conditions and reducing crop losses (Ott K H et al. (1996) J Mol Biol 263(2):359-368). The quality of the products may also be improved. Thus it is possible, for example, to increase the shelf life and storability of crop products by inactivating particular maturation genes. This was demonstrated, for example, by inactivating polygalacturonase in tomatoes (Hamilton A J et al. (1995) Curr Top Microbiol Immunol 197:77-89).
A basic prerequisite for transgenic expression of particular genes in plants is the provision of plant-specific promoters. Various plant promoters are known. It is possible to distinguish between constitutive promoters which enable expression in various parts of a plant, which is only slightly restricted in terms of location and time, and specific promoters which allow expression only in particular parts or cells of a plant (e.g. root, seeds, pollen, leaves, etc.) or only at particular times during development. Constitutive promoters are used, for example, for expressing “selection markers”. Selection markers (e.g. antibiotic or herbicidal resistance genes) permit filtering the transformation event out of the multiplicity of untransformed but otherwise identical individual plants.
Constitutive promoters active in plants have been written relatively rarely up to now. Promoters to be mentioned are the Agrobacterium tumefaciens, TR double promoter, the promoters of the vacuolar ATPase subunits or the promoter of a proline-rich wheat protein (WO 91/13991) and also the Ppc1 promoter Mesembryanthemum cryctallinum (Cushman et al. (1993) Plant Mol Biol 21:561-566).
The constitutive promoters which are currently the predominantly used promoters in plants are almost exclusively viral promoters or promoters isolated from Agrobacterium. In detail, these are the nopaline synthase (nos) promoter (Shaw et al. (1984) Nucleic Acids Res. 12(20):7831-7846), the mannopine synthase (mas) promoter (Comai et al. (1990) Plant Mol Biol 15 (3):373-381) and the octopine synthase (ocs) promoter (Leisner and Gelvin (1988) Proc Natl Acad Sci USA 85(5):2553-2557) from Agrobacterium tumefaciens and the CaMV35S promoter from cauliflower mosaic virus. The latter is the most frequently used promoter in expression systems with ubiquitous and continuous expression (Odell et al. (1985) Nature 313:810-812; Battraw and Hall (1990) Plant Mol Biol 15:527-538; Benfey et al. (1990) EMBO J. 9(69):1677-1684; U.S. Pat. No. 5,612,472). However, the CaMV 35S promoter which is frequently applied as constitutive promoter exhibits variations in its activity in different plants and in different tissues of the same plant (Atanassova et al. (1998) Plant Mol Biol 37:275-85; Battraw and Hall (1990) Plant Mol Biol 15:527-538; Holtorf et al. (1995) Plant Mol Biol 29:637-646; Jefferson et al. (1987) EMBO J. 6:3901-3907). A further disadvantage of the 35S promoter is a change in transgene expression in the case of an infection with cauliflower mosaic virus and its typical pathogenic variants. Thus, plants expressing the BAR gene under the control of the 35S promoter are no longer resistant after infection with the virus which typically occurs in nature (Al-Kaff et al. (2000) Nature Biotechnology 18:995-99).
From the range of viral promoters, the sugarcane bacilliform badnavirus (ScBV) which imparts an expression pattern similar to that of CamV has been described as an alternative to the CaMV 35S promoter (Schenk et al. (1999) Plant Mol Biol 39(6):1221-1230). The activity of the ScBV promoter was analyzed in transient expression analyses using various dicotyledonous plants, including Nicotiana tabacum and N. benthamiana, sunflower and oilseed rape, and monocotyledonous plants, here in the form of banana, corn and millet. In the transient analyses in corn, the ScBV promoter-mediated expression level was comparable to that of the ubiquitin promoter from corn (see below). Furthermore, the ScBV promoter-mediated rate of expression was assayed in transgenic banana and tobacco plants and displayed in both plant species essentially constitutive expression.
Common promoters for expressing selection markers in plants are especially the nos promoter, or else the mas promoter and ocs promoter, all of which have been isolated from Agrobacterium strains.
The use of viral sequences is often met with great reservations on the part of the consumer. These doubts are fed not least by studies which question the safety of the 35S CaMV promoter, owing to a possible horizontal gene transfer due to a recombination hot spot (Ho M W et al. (1999) Microbial Ecology in Health and Disease 11:194-197; Cummins J et al. (2000) Nature Biotechnology 18:363). It is therefore an aim of future biotechnological studies on plants to replace viral genetic elements by plant regulatory elements in order to keep as closely as possible to the plant system.
Owing to the prevailing doubts with regard to viral promoters, there are extensive efforts to replace said promoters by plant promoters. However, a promoter of plant origin, which is comparable to the viral elements, has not been described as yet.
What has been described, is a plant ubiquitin promoter from Arabidopsis thaliana (Callis et al. (1990) J Biol Chem 265:12486-12493; Holtorf S et al. (1995) Plant Mol Biol 29:637-747). Contrary to the findings in the articles mentioned, some studies revealed that the Arabidopsis ubiquitin promoter is unsuitable for expressing selection marker genes and that, for this reason, its general applicability must be called into question (see comparative examples 1 and 3).
The expression pattern mediated by the corn ubiquitin promoter has been described for the Ubi-1 and Ubi-2 promoters from corn (Christensen et al. (1992) Plant Mol Biol 18(4):675-689). While the Ubi-1 promoter has good expression activity in corn and other monocotyledonous plants, it exhibits in dicotyledonous tobacco plants only 10% of the activity which had been achieved in comparable experiments using the viral 35S promoter. It was furthermore shown that the corn Ubi-1 promoter is suitable for over expression of genes in monocotyledonous plant systems and, in addition, is sufficiently strong in order to mediate a herbicidal resistance via the expression of selection markers (Christensen and Quail (1996) Transgenic Res 5(3):213-218). The Ubi-1 promoter proved unsuitable for dicotyledonous expression systems.
A comparison of the organ specificity and strength of various constitutive promoters was carried out by Holtorf (Holtorf et al. (1995) Plant Mol Biol 29(4):637-646) on the basis of stably transformed Arabidopsis plants. The study comprised, inter alia, the CaMV35S promoter, the leaf-specific thionine promoter from barley and the Arabidopsis ubiquitin promoter (UBQ1). The CaMV35S promoter exhibited the highest rate of expression. On the basis of using an additional translational enhancer (TMV omega element), it was possible to increase the rate of expression of the promoter by a factor of two to three with unchanged organ specificity. The leaf-specific thionine promoter from barley was inactive in the majority of transformed lines, while the UBQ1 promoter from Arabidopsis resulted in medium rates of expression.
McElroy and colleagues reported a construct for transforming monocotyledonous plants, which is based on the rice actin 1 (Act1) promoter (McElroy et al. (1991) Mol Gen Genet. 231:150-160). Overall, it was concluded from the afore-described studies that the Act1 promoter-based expression vectors are suitable for controlling a sufficiently strong and constitutive expression of foreign DNA in transformed cells of monocotyledonous plants.
Another constitutive promoter which has been described is the promoter of an S-adenosyl-L-methionine synthetase (WO 00/37662). A disadvantage here is especially a dependence of the strength of expression on the methionine concentration (see WO 00/37662; FIG. 7).
WO 99/31258 describes chimeric constitutive plant promoters which are composed of various elements of various promoters with complementary expression patterns so that combination of individual tissue specificities additively results in a constitutive expression pattern.
Ferredoxin NADPH oxidoreductase (FNR) is a protein of the electron transport chain and reduces NADP+ to NADPH. Experiments in spinach using the spinach FNR promoter fused to the GUS gene hint at a light-inducible element in the FNR promoter (Oelmüller et al. (1993) Mol. Gen. Genet. 237:261-72). Owing to its function, a strictly leaf-specific expression pattern would have been expected for the promoter. Owing to the tissue-dependent expression pattern, the promoter would be poorly suited to expressing selection markers. Here, a selection in all tissue parts, if possible, is required in order to ensure efficient selection.
Owing to its function during photosynthesis, the promoter of the triose phosphate translocator (TPT) should be mainly leaf-specific. The cDNAs from potato (Schulz et al. (1993) Mol Gen Genet. 238:357-61), cauliflower (Fischer et al. (1997) Plant Cell 9:453-62), oilseed rape (WO 97/25346) and corn Kammerer B (1998) The Plant Cell 10:105-117) have been described. Kammerer et al. demonstrate that TPT mRNA expression in corn is strong in the leaves and the stamen. In contrast, no expression was observed in the stem or in the roots. Owing to the tissue-dependent expression pattern, the promoter would be poorly suited to expressing selection markers. Here, a selection in all tissue parts, if possible, is required in order to ensure efficient selection.
The “constitutive” promoters described in the prior art have one or more of the following disadvantages:
1. Inadequate homogeneity of expression:
The known “constitutive” promoters frequently display a different level of expression, depending on the type of tissue or cell. Moreover, the expression property is often highly dependent on the site of insertion into the host genome. As a consequence of this, the effects to be obtained by heterologous expression cannot be achieved to the same extent homogeneously in the plant. Under or over dosages may occur. This may have an adverse effect on plant growth or plant value.
2. Inadequate time profile:
The “constitutive” promoters known in the prior art often exhibit a nonconsistent activity during the development of a tissue. As a result, it is not possible, for example, to achieve desirable effects (such as selection) in the early phase of somatic embryogenesis which would be advantageous, especially here, due to the sensitivity of the embryo to in vitro conditions and stress factors.
3. Inadequate applicability to many plant species:
The “constitutive” promoters described in the prior art are often not active in the same way in all species.
4. If a plurality of expression cassettes with in each case the same “constitutive” promoter are present in an organism, interactions between said expression cassettes and even switching-off (gene silencing) of individual expression cassettes may occur (Mette et al. (1999) EMBO J. 18:241-248).
5. Promoters of viral origin may be influenced by virus infections of the transgenic plant and may then no longer express the desired property (Al-Kaff et al. (2000) Nature Biotechnology 18:995-99).
6. The public acceptance toward the use of promoters and elements from plant systems is higher than for viral systems.
7. The number of promoters suitable for expressing selection markers in plants is low and said promoters are usually of viral or bacterial origin.
8. Pollen/anther expression: The promoters mentioned (such as, for example, 35S CaMV) exhibit strong activity in the pollen or in the anthers. This may have disadvantageous effects on the environment. Thus, unspecific expression of Bacillus thuringiensis endotoxins resulted not only in the desired effect on feeding insects due to expression in the root but also, due to expression in the pollen, in considerable damage in the population of the Monarch butterfly which feeds predominantly on the pollen (Losey J E et al. (1999) Nature 399, 214).
An ideal constitutive promoter should have as many of the following properties as possible: (a) a gene expression which is as homogeneous as possible with regard to location and time, i.e. an expression in as many cell types or tissues of an organism as possible during the various phases of the developmental cycle. Furthermore, an efficient selection in differentiated cells (various callus phases) from a tissue culture and other developmental stages suitable for tissue culture is desired, (b) an applicability to various plant species, which is as broad as possible, and applicability to species in which it is not possible to achieve any expression using the “constitutive” promoters known to date, (c) to combine a plurality of transgenes in one plant, it is desirable to carry out a plurality of transformations in succession or to use constructs with a plurality of promoter cassettes, but without generating silencing effects due to the multiple use of identical regulatory sequences, (d) a plant origin in order to avoid problems of acceptance by the consumer and possible problems of future approval, and (e) secondary activities of a promoter in the anthers/pollen are undesirable, for example in order to avoid environmental damage, as discussed herein.